Retinoid signaling initiates when vitamin A is converted to retinoic acid (RA) which serves as a ligand for nuclear RA receptors that regulate gene expression and pattern formation during development of diverse organs including the limb buds, neural tube, and heart. Our understanding of how RA generation is regulated has been improved by discovery of three retinaldehyde dehydrogenases conserved in mouse and human that metabolize retinal to RA (RALDH1, RALDH2, and RALDH3). Null mutations of mouse Raldh2 almost totally eliminate RA synthesis and result in midgestation lethality with defects in the heart and hindbrain. Conditional rescue of Raldh2 mutants by limited maternal RA administration allows development to proceed (except for the forelimb buds) and results in the establishment of additional sites of RA synthesis linked to Raldh1 expression in the dorsal retina and to Raldh3 expression in the ventral retina, olfactory pit, and ureteric bud. Unexpectedly, conditionally RA-rescued Raldh2 mutants also possess novel sites of RA synthesis in the hindbrain, spinal cord, and heart that do not correspond to expression of Raldh1-3. Thus, additional enzymes perform RA synthesis in the neural tube and heart, plus the hindlimb bud receives RA from an unknown source. Administered RA rescues development of Raldh2 mutants not only directly, but also by stimulating additional tissue-specific RA generating enzymes that produce RA locally where it is needed. We are only at the beginning of the process in learning how embryonic tissues regulate the generation of RA, especially as additional enzymes need to be identified. Such information will be essential for development of stem cell-based treatments for disease. In order to induce organs to develop from stem cells, we need to have a thorough understanding of how the embryo performs this task. We hypothesize that embryonic retinoid signaling requires tissue-specific local RA synthesis and that some tissues utilize more than one enzyme, each with unique spatiotemporal expression patterns, to achieve this goal. We will use genetic approaches to detect RA in embryonic tissues and to examine the functions of potential RA generating enzymes. Before we can fully understand the impact of RA signaling we need to identify all the RA generating enzymes, clearly define when and where they function in embryonic tissues, and use null mutants to define the morphological consequences of their loss and the effect on downstream target genes. Specifically, conditionally RA-rescued Raldh2 mutants will be used to identify developmental processes in the limb buds, hindbrain, and spinal cord dependent upon RALDH2 or dependent upon other enzymes. Also, we will identify the novel RA-generating enzymes expressed in the neural tube and heart and begin genetic studies on them.